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Highlights
• High-throughput procedure for automated rapid and complete bisulfite conversion of DNA for
methylation analysis.
• Ready-to-use conversion reagent is added directly to DNA.
• High-yield, converted DNA is ideal for PCR, MSP, array, bisulfite and Next-Gen sequencing.
Contents
Product Contents ................................................. 1
Introduction to DNA Methylation ........................... 2
Product Description .............................................. 3
Product Specifications .......................................... 4
Reagent Preparation ............................................ 4
Protocol.............................................................5-6
Appendix .............................................................. 7
FAQs.................................................................... 8
Ordering Information ............................................ 9
List of Related Products ..................................... 10
Product Contents:
Instruction Manual 1 1 −
Note - Integrity of kit components is guaranteed for one year from date of purchase. Reagents are routinely tested on a
lot-to-lot basis to ensure they provide maximal performance and reliability.
* The Lightning Conversion Reagent is in a ready-to-use liquid format. The reagent should be stored tightly capped at
room temperature with minimum exposure to light.
** Add 288 ml of 100% ethanol to the 72 ml M-Wash Buffer concentrate before use.
***Two additional Collection Plates are provided as stands for the Conversion Plates during processing.
Use of Methylation Specific PCR (MSP) is protected by US Patents 5,786,146 & 6,017,704 & 6,200,756 & 6,265,171 and
International Patent WO 97/46705. No license under these patents to use the MSP process is conveyed expressly or by
implication to the purchaser by the purchase of this product.
Note - ™ Trademarks of Zymo Research Corporation. This product is for research use only and should only be used by
trained professionals. Some reagents included with this kit are irritants. Wear protective gloves and eye protection.
Follow the safety guidelines and rules enacted by your research institution or facility. Freedom EVO® is a registered
trademark and Te-Shake™ is a trademark of Tecan Group Ltd. Pyrosequencing® is a registered trademark of Biotage.
Product Description:
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Manual Automation
Manual Automated
Comparison of Manual vs. Automated Processing. Data show concentration, volume
Select Citations: and total yield for DNA samples across a 96-well plate. Half of the samples (rows A-D)
were processed manually. The other half of the samples “Automated” (rows E-H) were
1. Ehrich M, et al. Nuc. Acids processed using the Tecan – Freedom EVO® platform and a dedicated script.
Res. 2007; 35 (5): e29
Specifications:
Reagent Preparation:
Protocol:
2. Seal the plate with the provided film. Transfer the Conversion Plate to a thermal
cycler and perform the following steps:
5. Transfer the samples from the Conversion Plate into the Collection Plate
containing the M-Binding Buffer and MagBinding Beads. Mix by pipetting up and
down 3-6 times and, if available, vortexing at 1,300-1,500 rpm for 30 seconds (e.g.
Tecan - Te-Shake™).
Note: Transfer may be accomplished by either piercing or removing the cover foil on the Conversion Plate. If using
a Collection Plate as a stand for the Conversion Plate it may be necessary to secure the plates together by using
the tabs on the cover foil to prevent lifting of the Conversion Plate.
6. Let plate stand at room temperature for 5 minutes, then transfer plate to a magnetic
stand for an additional 5 minutes or until beads pellet and supernatant is cleared.
With the plate on the magnetic stand remove the supernatant and discard.
Note: Some beads will adhere to the sides of the well. Remove supernatant slowly to allow these beads to be
pulled to the magnet as the liquid level is lowered.
7. Remove the Collection Plate from the magnetic stand for this and each subsequent
buffer addition. Add 400 µl of M-Wash Buffer to the beads. Re-suspend the beads
by pipetting up and down or vortexing the plate at 1,300-1,500 rpm for 30 seconds.
Replace the plate on the magnetic stand for 3 minutes or until beads pellet.
Remove and discard supernatant.
Protocol (continued):
9. Add 400 µl of M-Wash Buffer to the beads. Re-suspend the beads by pipetting up
and down or vortexing for 30 seconds. Replace the plate on the magnetic stand for 3
minutes or until beads pellet. Remove and discard supernatant. Repeat this wash
step.
Note: Remove as much buffer as possible after final wash to aid in the drying of the beads.
10. Transfer the plate to a heating element at 55°C for 20-30 minutes to dry the beads
and remove residual M-Wash Buffer.
Alternatively, water or TE
Note: Beads will change in appearance from glossy black when still wet to a dull brown when fully dry. (pH ≥ 6.0) can be used for
elution if required for your
experiments.
11. Add 25 µl of M-Elution Buffer directly to the dried beads and pipette or vortex for 30
seconds to re-suspend. Heat the elution at 55°C for 4 minutes then transfer the
plate to the magnetic stand for 1 minute or until beads pellet. Remove the
supernatant and transfer to a clean Elution Plate.
Note: If beads are removed with the elution, slowly pippetting up and down one or two times will allow them to be
pulled to the magnet.
The DNA is ready for immediate analysis or can be stored at or below -20°C for later
use. For long term storage, store at or below -70°C. We recommend using 1-4 µl of
eluted DNA for each PCR, however, up to 25 µl can be used if necessary. The
elution volume can be > 25 µl depending on the requirements of your experiments,
but small elution volumes will yield higher DNA concentrations.
Automation Scripts:
Various automation scripts are available and can be obtained free of charge by
contacting Zymo Research at tech@zymoresearch.com. Include “Automation
Scripts” in the subject line and provide kit catalog number and the automation
platform desired in the email.
As most non-methylated cytosine residues are converted into uracil, the bisulfite-
ZymoTaq™ is a “hot start” treated DNA usually is AT-rich and has low GC composition. Non-specific PCR
DNA polymerase specifically amplification is relatively common with bisulfite treated DNA due to its AT-rich nature.
designed for the
amplification of bisulfite PCR using “hot start” polymerases is strongly recommended for the amplification of
treated DNA. (see page 10 bisulfite-treated DNA.
for details)
Q: Should the input DNA be dissolved in TE, water, or some other buffer prior to
its conversion?
A: Water, TE or modified TE buffers can be used to dissolve the DNA and do not
interfere with the conversion process.
Q: Why are there two different catalog numbers for the EZ-96 DNA Methylation-
Lightning™ Kit?
A: The two different catalog numbers are used to differentiate between the binding
plates that are included in the kit. Deep and shallow-well binding plates are available
to accommodate most rotors and microplate carriers. Below is a comparison of the
two binding plates.
Ordering Information:
Other…
ChIP DNA Clean & Clean and concentrate DNA from any reaction or “crude” preparation in 2 50 Preps. D5201 (uncapped column)
Concentrator™ min. A 6 µl minimum elution volume allows for highly concentrated DNA. 50 Preps. D5205 (capped column)
Designed for samples containing up to 5 µg of DNA.
Genomic DNA Clean & Genomic DNA clean-up in minutes. Unique spin column technology for 25 Preps. D4010
Concentrator™ recovery of ultra-pure large-sized DNA (100 bp to ≥200 kb) DNA from 100 Preps. D4011
any impure preparation (e.g., Proteinase K digestion).
ZymoTaq™ DNA ZymoTaq™ “hot start” DNA Polymerase is specifically designed for the 50 Rxns. E2001 (system)
Polymerase amplification of “difficult” DNA templates including: bisulfite-treated DNA 200 Rxns. E2002 (system)
for methylation detection. The product generates specific amplicons with
little or no by-product formation. Available either as a single buffer 50 Rxns. E2003 (premix)
premix or as a polymerase system with components provided separately. 200 Rxns. E2004 (premix)
Methylated-DNA IP Kit IP with a highly specific anti-5-methylcytosine monoclonal antibody. 10 Rxns. D5101
Designed for the enrichment of 5-methylcytosine-containing DNA from
any pool of fragmented genomic DNA for use in genome-wide
methylation analysis.
Services
Available for DNA Methylation and Hydroxymethylation at http://www.zymoresearch.com/services or inquire at
services@zymoresearch.com …powered by the latest Next-Gen Sequencing technologies!